SUMMARY/ABSTRACT Drugaddictionisacomplexdisease.Thereareavarietyofpotentialneuralsystemsandcircuits involved,someofwhicharephysiologicallyorepigeneticallyalteredthroughaddiction.Drugabuseand addictionweighheavilyontheU.S.throughhealthandcrime?relatedcosts,aswellasbroadersocietal issuesrelatedtosafety,family,educationandemployment.Inordertofullyunderstandthe physiologicalchangesintheaddictedbrainonacellularlevelwemusthavethepropertoolsforstudy. Thefirststeptowardcreatingthesetoolsistheestablishmentofaninvitrosystemofhuman dopaminergicneurons.Suchasystemwouldnotonlyenablecellular,molecular,andphysiological studiesintothemechanismsofaddictionbutalsoopenthedoortothescreeninganddevelopmentof smallmoleculesanddrugsthatcanpotentiallyblocktheonsetofchangesassociatedwithaddictionor evenreversethesechanges. Humanpluripotentstemcells(hPSC)canbedifferentiatedintopopulationsofneuronsenriched fordopaminergicneurons.However,currentprotocolsyieldlowpercentagesofdopaminergicneurons andarenotrobustandreproducible.Additionally,mostofthestemcell?to?dopaminergicneuron researchisfocusedonParkinson'sDiseaseandverylittleefforthasbeendedicatedtoderivingthe specifictypeofdopaminergicneuronsimplicatedinaddiction. TransfectionofsyntheticmRNAintocellsistechnologytofine?tunegeneexpression.This technologyhasbeenusedtoreprogramadultsomaticcellstopluripotentstemcellsandto transdifferentiateadultsomaticcellstoneuronsandothercelltypes.Weproposetodevelopnovel reagentsandprotocolsformRNA?mediateddirecteddifferentiationofhPSCtodopaminergicneurons relevantforaddictionresearch.Thisprojectwillyieldnovel,cell?specific,highefficiencymRNA transfectionreagentsandprotocols.Itwillresultinadetailedworkflowforthedirecteddifferentiation ofA10dopaminergicneuronsfromundifferentiatedhumaniPSC.Andfinally,itwillproduceaplatform systemforthestudyofmechanismsofdrugabuseatthecellularandmolecularlevel,whileallowing screeningassaystobeperformedonthissystem.Arealisticfutureapplicationofthisplatform(PhaseII) isthecreationofiPSCfromcellstakenfromaddictedindividualscarryingidentifiedgeneticvariants, identifiedtoberiskfactorsforaddictivebehavior.Thesepatient?specificiPSCcanthenbedifferentiated toA10DAneuronsandstudiedforpossibleinsightsintohowthesegeneticvariationsaffectthe developmentofaddictivebehavior,amongotherthings.